Corresponding author: Jessica L. Reiner 1

National Institute of Standards and Technology 2

331 Fort Johnson Rd. 3

Charleston, SC 29412 4

Phone: 843-725-4822 5

Fax: 843-762-8742 6

Email: jessica.reiner@nist.gov 7

https://ntrs.nasa.gov/search.jsp?R=20160005296 2018-06-22T06:08:34+00:00Z

Perfluorinated Alkyl Acids in plasma of American alligators (Alligator mississippiensis) 8

from Florida and South Carolina 9

Jacqueline T. Bangma†, John A. Bowden‡, Arnold M. Brunell§, Ian Christieǁ, Brendan 10

Finnell#, Matthew P. Guillette†, Martin Jones††, Russell H. Lowers‡‡, Thomas R. 11

Rainwater§§, Jessica L. Reiner‡, Philip M. Wilkinsonǁǁ, and Louis J. Guillette Jr.†## 12

†Medical University of South Carolina, Department of Obstetrics and Gynecology, 221 Fort 13

Johnson Road, Charleston, SC29412 USA 14

‡National Institute of Standards and Technology, Chemical Sciences Division, Hollings Marine 15

Laboratory, 331 Fort Johnson Road, Charleston, SC 29412 USA 16

§Florida Fish and Wildlife Conservation Commission, 601 W. Woodward Ave, Eustis, FL 17

32726, United States 18

ǁGrice Marine Laboratory, College of Charleston, 205 Fort Johnson Road, Charleston, SC 29412 19

USA 20

#Illinois Wesleyan University, 1312 Park Street, Bloomington, IL 61701 USA 21

††College of Charleston, Department of Mathematics, 66 George Street, Charleston, SC 29424 22

USA 23

‡‡Integrated Mission Support Service (IMSS), Titusville, FL USA 24

§§Baruch Institute of Coastal Ecology and Forest Science, Clemson University, P.O. Box 596, 25

Georgetown, South Carolina 29442, USA 26

ǁǁTom Yawkey Wildlife Center, 407 Meeting Street, Georgetown, South Carolina 29440, USA 27

##Author passed away August 6, 2015. 28

29

Abstract: 30

31

This study aimed to quantitate fourteen perfluoroalkyl acids (PFAAs) in 125 adult 32

American alligators at twelve sites across the southeastern US. Of those fourteen PFAAs, nine 33

were detected in 65 % - 100 % of the samples: PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTriA, 34

PFTA, PFHxS, and PFOS. Males (across all sites) showed significantly higher concentrations of 35

four PFAAs: PFOS (p = 0.01), PFDA (p = 0.0003), PFUnA (p = 0.021), and PFTriA (p = 0.021). 36

Concentrations of PFOS, PFHxS, and PFDA in plasma were significantly different among the 37

sites in each sex. Alligators at Merritt Island National Wildlife Refuge and Kiawah Nature 38

Conservancy both exhibited some of the highest PFOS concentrations (medians 99.5 ng/g and 39

55.8 ng/g respectively) in plasma measured to date in a crocodilian species. A number of positive 40

correlations between PFAAs and snout-vent length (SVL) were observed in both sexes 41

suggesting PFAA body burdens increase with increasing size. In addition, several significant 42

correlations among PFAAs in alligator plasma may suggest conserved sources of PFAAs at each 43

site throughout the greater study area. This study is the first to report PFAAs in American 44

alligators, reveals potential PFAA hot spots in Florida and South Carolina, and provides and 45

additional contaminant of concern when assessing anthropogenic impacts on ecosystem health. 46

Keywords: PFOS PFHxS alligator crocodilians plasma 47

48

INTRODUCTION 49

Despite being manufactured for over 50 years [1], it wasn’t until 2000 that the class of 50

chemicals known as perfluoroalkyl acids (PFAAs) entered the scientific spotlight as a major 51

environmental contaminant of concern [2]. The two most commonly known PFAAs, 52

perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), were first produced by 53

3M in 1948 [1] and 1947, respectively, the latter of which was subsequently purchased by 54

DuPont in 1951 [3]. A variety of new PFAAs have steadily been introduced to the market since 55

the introduction of these first PFAAs. Structurally, PFAAs can widely vary, but as a whole, they 56

typically consist of carbon chains of varying length (linear and branched isomers), an acid 57

functional group, and hydrogen atoms substituted with fluorine atoms [4]. The carbon–fluorine 58

bonds are the unique feature of PFAAs and provide chemical and thermal stability [5]. Two well-59

studied families of PFAAs are carboxylic acids and sulfonic acids [2, 6]. 60

The usage of PFAAs has become widespread since the introduction of these chemicals in 61

the 1940s, largely because they exhibit unique surfactant properties that make them attractive 62

components for many consumer-related products, such as non-stick pans, water repellant 63

surfaces, hair products, plastics, and lubricants [2], as well as firefighting products known as 64

aqueous film-forming foams (AFFF) [7]. Active manufacturing and use of certain PFAAs, like 65

PFOS and PFOA, have largely ceased owing to a voluntary phase-out by industry. Current 66

production of fluorinated chemicals includes shorter chained carboxylic and sulfonic acid 67

substitutes, like perfluorobutanesulfonic acid (PFBS) and perfluorobutyric acid (PFBA), 68

respectively [8]. In addition, precursor chemicals that have a non-fluorinated structural 69

component attached to a perfluorinated chain may be amenable to microbial or chemical 70

transformation and have the potential to degrade into perfluorinated carboxylic and sulfonic 71

acids over time [9]. 72

The same properties that make PFAAs commercially valuable (e.g. the highly stable 73

carbon fluorine bonds) also enable them to persist in the environment by resisting chemical, 74

microbial, and photolytic degradation. However, unlike the more lipophilic environmental 75

contaminants such as organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and 76

brominated flame retardants (PBDEs) that are sequestered in adipose tissue, PFAAs accumulate 77

in the blood and blood-rich organs, such as the liver [10, 11]. Conversely, like OCPs, PCBs, and 78

PBDEs, PFAAs have also been shown to bioaccumulate and biomagnify in food webs [6]. 79

Increasing PFAA chain length has been shown to correlate with an increasing ability to 80

bioaccumulate [12], and the greatest PFAA concentrations detected in wildlife have been in 81

species occupying high trophic positions [13]. Because PFAAs are bioaccumulative and often 82

observed in higher concentrations in fish-eating marine species [13], humans who consume more 83

fish in their diet may be at higher risk of PFAA exposure than those who consume less fish [14]. 84

Animal studies reveal a wide range of PFAA-related effects that include alterations in 85

liver physiology and serum cholesterol, as well as resulting hepatomegaly, wasting syndromes, 86

neurotoxicity, immunotoxicity [15-17]. In addition, PFAAs have also been mentioned as possible 87

obesogens due to their interaction with peroxisome proliferator activated receptors (PPAR) 88

receptors [18]. However, although species-specific variations in PFAA excretion rates have been 89

observed [2], the actual mechanism(s) of action of PFAA toxicity is not well understood. 90

Few reports exist on PFAA distribution and body burdens in North American wildlife, 91

and studies of PFAAs in wild reptiles and amphibians have been limited almost exclusively to 92

frogs and sea turtles [19]. Globally, only two studies have examined PFAAs in crocodilians [20, 93

21]. Because of their high trophic status, long life span, and high site fidelity, crocodilians are 94

attractive study species for ecotoxicological investigations, particularly those involving exposure 95

and accumulation of persistent environmental contaminants [23-25]. As such, studies examining 96

PFAAs in crocodilians can provide insight into exposure and potential effects in focal species as 97

well as identify potential hot spots of PFAA contamination. 98

In this study, we examined PFAA concentrations in plasma of wild American alligators 99

(Alligator mississippiensis) from 12 sites in Florida and South Carolina (Figure 1). Because 100

factors such as sex, body size, and location may influence PFAA concentrations in alligators, the 101

relationships between PFAA body burdens and these parameters were also examined. 102

MATERIALS AND METHODS 103

Study Area 104

American alligator plasma samples (n = 125) were collected between 2012 and 2015, as 105

part of multiple ongoing projects examining the biology and ecotoxicology of alligators in 106

Florida and South Carolina [22-24]. In South Carolina, alligator blood samples were collected 107

from the following sites (in order of North to South): Tom Yawkey Wildlife Center (YK, n = 108

10), Kiawah Island (KA, n =10), and Bear Island Wildlife Management Area (BI, n = 10) 109

(Figure 1, Table S1). In Florida, samples were collected from the following sites (in order from 110

North to South): Lochloosa Lake (LO, n = 10), Lake Woodruff (WO, n = 10), Lake Apopka (AP, 111

n = 10), Merritt Island National Wildlife Refuge (MI, n = 15), St. John River (JR, n = 10), Lake 112

Kissimmee (KS, n = 10), Lake Trafford (TR, n =10), Everglades Water Conservation Area 2A 113

(2A, n = 10), and Everglades Water Conservation Area 3A (3A, n = 10) (Figure 1, Table S1). 114

Sample collection 115

Immediately following capture, a blood sample was collected from the post-occipital 116

sinus of the spinal vein of each animal using a sterile needle and syringe [22-24]. Whole blood 117

samples were then transferred to 8 mL lithium-heparin Vacutainer blood collection tubes (BD, 118

Franklin Lakes, NJ), stored on ice in the field, and later centrifuged at 2500 rpm at 4 C for 10 119

min to obtain plasma, which was stored at -80 °C until analysis. Snout-vent length (SVL) was 120

measured for each animal, and sex was determined by cloacal examination of the genitalia [25]. 121

The National Institute of Standards and Technology (NIST) Standard Reference 122

Materials (SRM) 1958 Organic Contaminants in Fortified Human Serum was used as a control 123

material during PFAA analysis. The freeze-dried human serum SRM 1958 was reconstituted 124

with deioinized water according to the instructions on the Certificates of Analysis 125

(www.nist.gov/srm/) and analyzed alongside collected alligator plasma. 126

Chemicals 127

Calibration solutions were created by combining two solutions produced by the NIST 128

Reference Materials (RMs) 8446 Perfluorinated Carboxylic Acids and Perfluorooctane 129

Sulfonamide in Methanol and RM 8447 Perfluorinated Sulfonic Acids in Methanol. Together, 130

the solution contained 15 PFAAs as follows: PFBA, perfluoropentanoic acid (PFPeA), 131

perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorononanoic 132

acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), 133

perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTriA), perfluorotetradecanoic 134

acid (PFTA), PFBS, perfluorohexanesulfonic acid (PFHxS), PFOS, and 135

perfluorooctanesulfonamide (PFOSA). 136

Internal standards (IS) were purchased from Cambridge Isotope Laboratories (Andover, 137

MA), RTI International (Research Triangle Park, NC), and Wellington Laboratories (Guelph, 138

Ontario) to create an internal standard (IS) mixture comprised of eleven isotopically labeled 139

PFAAs, and they were as follows: [13C4]PFBA, [13C2]PFHxA, [13C8]PFOA, [13C9]PFNA, 140

[13C9]PFDA, [13C2]PFUnA, [13C2]PFDoA, [18O2]PFBS, [18O2]PFHxS, [13C4]PFOS, and 141

[18O2]PFOSA. 142

Sample preparation 143

Samples were extracted using a method previously described in 2011 by Reiner et al. 144

[26]. Approximately 1 mL of each alligator plasma sample and SRM 1958 aliquots were thawed 145

and gravimetrically weighed. All samples were then spiked with the IS mixture (approximately 146

600 µL) and gravimetrically weighed. After brief vortex-mixing and 90 min of equilibration, 4 147

mL of acetonitrile were used to extract the PFAAs from each sample. After sonication and 148

centrifugation, the supernatant was removed from all samples. The collected supernatant was 149

then solvent exchanged to methanol and further purified using an Envi-carb cartridge (Supelco, 150

Bellefonte, PA). Resulting extracts were evaporated to 1 mL using nitrogen gas prior to being 151

analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 152

Samples were analyzed using an Agilent 1100 HPLC system (HPLC; Santa Clara, CA) 153

coupled to an Applied Biosystems API 4000 triple quadrupole mass spectrometer (Applied 154

Biosystems, Foster City, CA) with electrospray ionization in negative mode. Samples were 155

examined by LC using an Agilent Zorbax Eclipse Plus C18 analytical column (2.1 mm x 150 156

mm x 5µm). A ramping LC solvent gradient was employed using methanol and de-ionized water 157

both containing 20 mmol/L ammonium acetate [26]. Two multiple reaction monitoring (MRM) 158

transitions for each PFAA were monitored to ensure no interferences with measurements, one 159

MRM was employed for quantitation and the other one was used for confirmation [26]. 160

Quality control 161

All alligator plasma samples were processed alongside quality control material NIST 162

SRM 1958 and blanks to determine the accuracy and precision of the method. The PFAA levels 163

of SRM 1958 processed during our extraction had to agree with previously established values 164

reported on the Certificate of Analysis (CoA). In addition, compounds were considered to be 165

above the reporting limit (RL) if the mass of an analyte in the sample was greater than the mean 166

plus three standard deviations of all blanks. 167

Statistical Methods 168

All statistical analyses were performed using IBM SPSS statistic 22 (Armonk, NY: IBM 169

Corp.). Statistical tests were performed for the compounds detected in greater than 75 % of the 170

samples: PFNA, PFDA, PFUnA, PFDoA, PFTriA, PFTA, PFHxS, and PFOS. Unlike many 171

environmental studies where PFOA is the second highest burden PFAA measured, PFOA was 172

detected at much lower concentrations than many of the above PFAAs and was detected in only 173

65 % of the samples. With a full one third of PFOA measurements falling below the limit of 174

detection (LOD), PFOA was excluded from statistical analysis along with the remaining 175

chemicals (PFHpA, PFHxA, PFPeA, PFBS, and PFBA) that were detected in less than 2% of the 176

samples. For those PFAAs included in statistical analysis, compounds less than the LOD were 177

set equal to half the LOD prior to running the statistical tests [27]. 178

Sex-based differences of PFAAs in Florida and South Carolina were investigated using 179

univariate analysis of variance with log normally distributed concentration values, and a 180

Friedman’s test was used for the PFAAs with non-normally distributed concentration values. Site 181

was set as the nuisance factor, sex as the treatment, and PFAA burden as the dependent variable. 182

These tests simulated a randomized block design for the collected data. Other parametric tests 183

employed for data analysis of sex-based differences, on a site by site basis and analysis of site 184

differences for PFAA levels, included a t-test and one-way ANOVA when data were normal or 185

log-normal and Friedman rank test, Mann-Whitney U test and Kruskal Wallis test when data 186

remained non-normal following log transformation. Pearson correlation and Spearman 187

correlation were used when applicable for correlative measures. 188

RESULTS AND DISCUSSION 189

In this study, we collected a total of 125 plasma samples from alligators at multiple sites 190

in Florida and South Carolina to examine PFAA concentrations in animals from different 191

localities. Of the 15 PFAAs included in our analysis, all samples contained at least six. The 192

following five PFAAs were detected in every plasma sample analyzed (in order of highest 193

overall burden to lowest overall burden, among all sites): PFOS (median 11.2 ng/g, range 1.36–194

452 ng/g), PFUnA (median 1.58 ng/g, range 0.314–18.4 ng/g), PFDA (median 1.20 ng/g, range 195

0.169–15.1 ng/g), PFNA (median 0.528 ng/g, range 0.155–1.40 ng/g), and PFHxS (median 0.288 196

ng/g, range 0.057–23.3 ng/g) (Table 1 and Table S2). PFDoA, PFTriA, PFTA, and PFOA were 197

also detected frequently in alligator plasma samples (over 96 %, 94 %, 75 %, and 65 %, 198

respectively): PFDoA (median 0.363 ng/g, range <0.009–7.27 ng/g), PFTriA (median 0.416 199

ng/g, range <0.026–2.60 ng/g), PFTA (median 0.050 ng/g, range <0.008–1.38 ng/g), and PFOA 200

(median 0.064 ng/g, range <0.008–0.412 ng/g) (Table 1 and Table S2). The nine PFAAs 201

commonly measured over the LOD resulted in unique fingerprints for each site (Figure S1), 202

which are discussed below. The shorter chain PFAAs (PFHpA, PFHxA, PFPeA, PFBS, and 203

PFBA) were detected infrequently (< 2 % of the samples) and therefore were not included in any 204

statistical analysis. 205

Sex differences 206

Across all sites, male alligators exhibited significantly higher concentrations of several 207

PFAAs in plasma compared to females as a group: PFOS (p = 0.01, Figure 2), PFDA (p = 208

0.0003, Figure S2), PFUnA (p = 0.021, Figure S2), and PFTriA (p = 0.021, Figure S2). 209

However, at some sites, PFAA concentrations were significantly higher in females (e.g., PFOS 210

at AP, PFUnA at KA). 211

In a population of captive Chinese alligators (Alligator sinensis), Wang et al. [21] found 212

the highest PFAA concentrations in serum to be that of PFUnA rather than PFOS, the PFAA 213

with the highest concentrations in our study. However, similar to our study, male Chinese 214

alligators contained significantly higher concentrations of PFOS and PFUnA compared to 215

females. Wang et al. [22] did not find a sex-based difference for PFDA in Chinese alligators. It is 216

possible that sex-based differences observed for certain PFAAs in alligators is due to a 217

differential clearance of these contaminants between males and females, as has been observed in 218

rats [28], mice [29], and other mammals [30]. It is also possible females may offload PFAAs 219

during oviposition, reducing their PFAA body burden compared to males at the same locality. 220

This possibility is supported by studies reporting measurable concentrations of PFAAs in eggs 221

of herring gulls (Larus argentatus) [31] and Nile crocodiles (Crocodylus niloticus) [20], 222

confirming maternal transfer of PFAAs in oviparous species. Sex-specific differences in PFAA 223

concentrations may also be the result of differential habitat use by adult males and females, a 224

phenomenon common among crocodilians [32-35]. In such cases, differences are prey 225

availability and contamination between and among habitats within a site could result in different 226

PFAA exposures in males and females. 227

Because no sex-specific differences in PFOA, PFNA, PFHxS, PFDoA, and PFTA 228

concentrations were observed among sampling localities in this study, sex-based differences 229

were examined on a site-by-site basis (Table S3). Overall, only a few sites exhibited sex-based 230

differences for these 5 PFAAs (Figure S3). At LO, male alligators had significantly higher PFNA 231

(p = 0.016), PFTA (p = 0.032), and PFDoA (p = 0.032) plasma concentrations compared to 232

females, and at MI males had significantly higher PFOA (p = 0.047) plasma concentrations than 233

females. Interestingly, PFHxS was the only PFAA (of the five investigated on a site by site basis) 234

for which females exhibited significantly higher plasma concentrations (YK, p = 0.008, TR, p = 235

0.008) when compared to males (Figure S3). It is important to note our examination of sex-based 236

differences in PFAA concentrations may have been influenced by small samples sizes, as in 237

almost all cases only five males and five females were sampled per site. 238

Site differences 239

Because sex-based differences in PFAA concentrations were observed among alligator 240

plasma samples, site differences were determined separately for males and females. Of the nine 241

detected PFAAs, all of them displayed at least some minor site differences. The PFAAs that 242

displayed the most notable site differences (the most number of statistically significant groups 243

between the 12 sites) were PFOS, PFDA, and PFHxS. Of those three, PFOS exhibited the 244

greatest statistical difference across sites (Figure 3). This is likely due to the fact that PFOS is 245

generally the most ubiquitous PFAA in the environment. When combining both sexes, 246

concentrations of PFOS in alligator plasma ranged from 1.36 ng/g - 452 ng/g. For male 247

alligators only, concentrations of PFOS in plasma ranged from 1.57 ng/g to 452 ng/g. PFOS 248

concentrations were highest at MI (median 106 ng/g) and KA (median 56.4 ng/g). MI males 249

exhibited significantly higher PFOS concentrations compared to all other sites with the exception 250

of KA. In addition, the individual alligator with the highest overall 251

PFOS concentration measured in this study (452 ng/g plasma) was from MI. After MI, 252

males from South Carolina (KA, YK, and BI) exhibited higher PFOS concentrations than Florida 253

males, with the exception of WO. Some of the lowest PFOS concentrations observed in males in 254

this study were measured at sites 2A, 3A, LO, and JR. 255

For female alligators, PFOS concentrations in plasma ranged from 1.36 ng/g - 206 ng/g. 256

Similar to males, females at sites MI (median 85.5 ng/g) and KA (median 51.3 ng/g) exhibited 257

significantly higher PFOS concentrations compared to the other sites examined, and the 258

individual female with highest PFOS concentration was from MI (206 ng/g plasma). After MI 259

and KA, females from the two other South Carolina sites (YK and BI) exhibited higher PFOS 260

concentrations than males from Florida, with the exception of WO and AP. Some of the lowest 261

PFOS concentrations observed in females in this study were measured at sites 2A, 3A, LO, JR, 262

and TR. 263

The concentrations of PFHxS detected in alligator plasma in this study exhibited a similar 264

trend to PFOS across sites, but on a reduced scale (Table S4). Male and female PFHxS plasma 265

concentrations ranged from 0.0566 ng/g – 23.3 ng/g throughout the sampling sites the entire 266

southeast. For males, PFHxS plasma concentrations ranged from 0.057 ng/g – 23.3 ng/g. Males 267

from MI (median 3.95 ng/g) had significantly higher PFHxS concentrations than any other site 268

examined, and the individual male with highest PFHxS concentration was from site MI (23.3 269

ng/g). Males from KA and KS followed closely, but were still statistically grouped with other 270

sites (AP, WO, and BI). The lowest PFHxS concentrations in males were typically measured at 271

sites 2A, 3A, LO, and TR. For female alligators, PFHxS concentrations in plasma ranged from 272

0.069 ng/g – 10.0 ng/g. Like males, MI females exhibited significantly higher PFHxS 273

concentrations than all other sites. Females at KA and KS had the next highest concentrations but 274

were still statistically grouped with other sites (AP, WO, and YK). The lowest PFHxS 275

concentrations in females were typically observed at sites 2A, 3A, and LO. 276

PFDA had a unique signature across the sampling sites, one that varied from the patterns 277

observed for plasma PFOS and PFHxS concentrations (Table S4). PFDA plasma concentrations 278

ranged from 0.169 ng/g - 15.1 ng/g for all sites examined. For male alligators, PFDA 279

concentrations ranged from 0.498 ng/g – 15.1 ng/g. KA males had significantly higher PFDA 280

concentrations overall (median 6.21 ng/g) compared to all sites, with the exception of YK 281

(median 6.20 ng/g). YK males exhibited the next highest PFDA concentrations, but these were 282

not significantly different from those detected in WO males (median 2.02 ng/g). Males at many 283

of the remaining sites had similarly low concentrations of PFDA. Overall, LO males (median 284

0.792 ng/g) had some of the lowest PFDA concentrations of all the sampling sites. For female 285

alligators, PFDA plasma concentrations ranged from 1.69 ng/g – 14.3 ng/g. The two sites with 286

the highest (statistically significant) PFDA concentrations in females were also in South 287

Carolina: KA (median 6.32 ng/g) and YK (median 5.55 ng/g). PFDA concentrations at BI 288

(median 1.18 ng/g) and WO (median1.84) followed closely behind, but were not significantly 289

different from the other sites sampled. Like males, LO females (median 0.501 ng/g) had some of 290

the lowest PFDA concentrations across all sites. 291

Overall, male and female alligators from both MI and KA exhibited some of the highest 292

PFOS concentrations measured to date in a crocodilian species (median PFOS concentrations in 293

plasma: MI males = 106 ng/g, MI females = 85.5 ng/g. KA males = 56.4 ng/g, KA females = 294

51.3 ng/g). In comparison, the mean PFOS concentration in serum from captive Chinese 295

Alligators was 28.7 ng/mL (28.0 ng/g) [21]. In other reptiles, Kemp’s ridley sea turtles 296

(Lepidochelys kempii) and loggerhead sea turtles (Caretta caretta) along the coast of South 297

Carolina, Georgia, and Florida exhibited median PFOS plasma concentrations of 41900 pg/ml 298

(40.9 ng/g) and 5450 pg/ml (53.2 ng/g), respectively [27]. The high concentrations of PFOS and 299

PFHxS detected in male and female alligators at MI may be related to the aeronautic facilities 300

located in and around MI that comprise up a large part of Florida’s Kenndey Space Center. The 301

use of AFFF is not uncommon at Kennedy Space Center, and may contribute to PFAAs in the 302

surrounding environment. Historically, AFFF have been shown to contain PFAAs, such as PFOS 303

and PFHxS, as well as a number of other propriety PFAA mixtures [7] and can be resistant to 304

remediation [9]. PFAAs have been measured in firefighters [36], wildlife [6], and downstream of 305

their use [37]. Potential sources of PFOS and PFHxS at KA are more speculative. In addition, it 306

should be noted with the exclusion of MI, alligators from the South Carolina sites (BI, YK, and 307

KA) had some of the highest PFOS concentrations compared to the Florida sites. In Florida, WO 308

exhibited mid to high concentrations of PFOS, PFHxS, and PFDA compared to other sampled 309

sites. For many years, WO has been used as a reference site for multiple studies on alligator 310

ecotoxicology due to its relatively low concentrations of organochlorine contaminants, such as 311

DDT, its metabolites, and other OCPs [38]. The results of the present study obviously indicate 312

WO would not be a suitable reference site for future studies involving PFAAs. In contrast to 313

WO, sites 2A and 3A, which are located in the Everglades, exhibited some of the lowest 314

concentrations of PFOS and PFHxS measured in Florida. Interestingly, while PFAA 315

concentrations appear to be relatively low, adult alligators at these sites have been reported to 316

contain some of the highest mercury concentrations in Florida and throughout the range of the 317

species [24, 39, 40]. 318

Correlations 319

For all alligators included in the study, SVL was uniform across sites for males and 320

nearly uniform across sites for females (Figure S4). Thus, data from all sites were combined 321

within each sex to investigate relationships between PFAA burden and alligator SVL. 322

Correlations comparing both male SVL to PFAAs and female SVL to PFAAs resulted in a 323

number of significant positive correlations (Table 2). Overall, females exhibited higher 324

correlation coefficients between PFAA concentration and SVL when compared to the males. The 325

highest correlation coefficients for females were with PFTriA, which explained 57.0 % of the 326

variation, followed closely by PFOS, which explained 55.1 % of the variation. In contrast, the 327

highest correlation coefficients for male SVL and PFAA concentration was PFUnA, which 328

explained 35.5 % of the variation, followed closely by PFOS, which explained 33.1 % of the 329

variation. Collectively, these data suggest concentrations of some PFAAs in adult American 330

alligators increase with increasing body size in both males and females. Conversely, Wang et al. 331

[21] found that PFAA (PFUnA, PFDA, and PFNA) concentration decreased with increasing 332

body size (total length). These observed differences between American and Chinese alligators 333

may be the result of interspecific differences in food consumption, growth rates, and body size 334

[41] as well as in toxicodynamics and toxicokinetics of PFAAs. In addition, differences in diet 335

and numerous environmental variables between wild (this study) and captive [22] alligators may 336

influence growth and body burdens of PFAAs. Finally, differences in sample types (plasma vs 337

serum) between the two studies may have had some effect on PFAA-body size relationships. 338

With all sites combined for each sex, significant correlations were observed between 339

different PFAAs measured in plasma, suggesting somewhat similar sources of PFAA 340

contamination across the sampling localities. The varying levels of PFAA contamination from 341

site to site are likely due to varying distances from these potential PFAA sources. Some 342

correlative relationships between the PFAAs were stronger than others (Table 3). Of all the 343

PFAAs, correlations between PFUnA and PFDoA for male (p < 0.01, r = 0.920) and female (p < 344

0.01, r = 0.938) alligators across the sites were the most highly significant relationships observed 345

in this study. 346

CONCLUSIONS 347

This study is the first to quantitate PFAA concentrations in American alligators and one 348

of the few studies to quantitate PFAAs in crocodilians [20, 21]. All alligator samples (n = 125) 349

contained at least six quantifiable PFAAs: PFOS (median 11.2 ng/g, range 1.36–452 ng/g), 350

PFUnA (median 1.58 ng/g, range 0.314–18.4 ng/g), PFDA (median 1.20 ng/g, range 0.169–15.1 351

ng/g), PFNA (median 0.528 ng/g, range 0.155–1.40 ng/g), and PFHxS (median 0.288 ng/g, range 352

0.057–23.3 ng/g). Our findings support sex-based differences in PFOS and PFUnA 353

concentrations previously observed in captive Chinese alligators [21], while demonstrating 354

opposite relationships between PFAA concentration and body size exist for American (wild) and 355

Chinese (captive) alligators A high number of significant PFAA to PFAA correlations suggest 356

common point sources throughout the samplimg sites in Florida and South Carolina. This study 357

also reveals potential hot spots for various PFAAs (e.g., PFOS at KA and MI) that warrant 358

further investigation. and provides another contaminant of concern to be combined with 359

organochlorines, metals, and others when assessing overall anthropogenic impacts on ecosystem 360

health. 361

Acknowledgments -This project was completed as part of the 2014 Fort Johnson 362

Research Experience for Undergraduates (REU) program, operated by the College of Charleston, 363

and supported by the U.S. National Science Foundation under Award Number DBI-1359079, 364

and as part of the 2015 Summer Undergraduate Research Program, operated by the Medical 365

University of South Carolina and supported by the Marine Biomedicine and Environmental 366

Sciences (MBES) Director’s Fund. Finally, we would like to thank our collaborators: South 367

Carolina Department of Natural Resources, Tom Yawkey Wildlife Center, Florida Fish and 368

Wildlife Conservation Commission, Kiawah Nature Conservancy, and Integrated Mission 369

Support Service, Titusville, FL for assistance and support during this project. This work would 370

not have been possible without the passion, guidance, and support of Dr. Louis J. Guillette Jr.. 371

His mentorship and friendship will be greatly missed. 372

Disclaimer - Certain commercial equipment or instruments are identified in the paper to 373

specify adequately the experimental procedures. Such identification does not imply 374

recommendations or endorsement by the NIST nor does it imply that the equipment or 375

instruments are the best available for the purpose. 376

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486 487

FIGURE LEGENDS 488 489

Figure 1. Map showing the twelve sites from which American alligators (Alligator 490

mississippiensis) were sampled in this study (n = 125) during the years 2012 – 2015. 491

Collection sites are listed in decreasing latitude. 492

493

Figure 2. Mean (±SD) PFOS concentrations (log; ng/g) in male and female American alligators 494

(Alligator mississippiensis) sampled at multiple sites in Florida and South Carolina. 495

Samples are listed from left to right in decreasing latitude. Refer to figure 1 for site 496

abbreviations. 497

498

Figure 3. Mean (±SD) PFOS concentrations (log; ng/g) in (A) male and (B) female American 499

alligator (Alligator mississippiensis) plasma from multiple sites in Florida and South 500

Carolina. Letters above bars represent statistically significant differences between groups 501

(p < 0.05).Samples are listed from left to right in decreasing latitude. Refer to figure 1 for 502

site abbreviations.503

Table 1. Alligator perfluoroalkyl acid (PFAA) plasma concentrations (ng/g wet mass) at 12 sites from Florida and South Carolina:

Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA),

perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTriA), perfluorotetradecanoic acid (PFTA), perfluorohexanesulfonic

acid (PFHxS), and perfluorooctane sulfonate (PFOS).

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

PFOA <0.096b-0.152 0.126 7 <0.008

b-0.193 < 0.100 3 0.028-0.298 0.126 10 <0.008

b-0.142 0.104 9 <0.008

b-0.132 0.071 9 <0.096

b-0.412 0.155 7

PFNA 0.251-1.40 0.648 10 0.155-1.14 0.472 10 0.446-1.38 1.19 10 0.275-1.18 0.642 10 0.328-1.19 0.676 10 0.298-1.10 0.611 15

PFDA 0.169-2.44 1.12 10 0.998-3.21 1.57 10 3.72-13.6 6.26 10 0.417-3.15 1.26 10 0.238-1.00 0.615 10 0.395-3.50 1.02 15

PFUnA 0.614-3.39 1.65 10 1.05-5.02 2.32 10 1.87-7.53 3.93 10 0.314-2.47 1.03 10 0.580-1.56 1.03 10 0.844-5.45 1.82 15

PFDoA <0.157b-0.831 0.315 9 0.231-1.88 0.559 10 1.32-7.27 3.05 10 <0.009

b-0.382 0.147 9 0.105-0.309 0.182 10 <0.543

b-1.07 0.418 14

PFTriA 0.189-1.00 0.450 10 <0.070b-1.83 0.674 9 0.420-2.60 0.919 10 0.122-0.677 0.251 10 0.181-0.580 0.309 10 <0.026

b-1.42 0.654 14

PFTA <0.080b-0.194 0.049 7 <0.081

b-0.733 0.095 7 0.198-1.38 0.476 10 <0.009

b-0.104 0.025 9 <0.008

b-0.060 0.018 7 <0.080

b-0.257 0.076 6

PFHxS 0.166-0.449 0.332 10 0.077-0.824 0.304 10 0.313-1.86 0.620 10 0.338-1.50 0.505 10 0.069-0.201 0.093 10 0.684-23.3 3.83 15

PFOS 1.98-15.8 11.4 10 10.0-44.9 19.5 10 38.4-98.2 55.8 10 6.51-25.1 12.2 10 2.19-6.16 4.21 10 38.6-452 99.5 15

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

Range Median n > RLa

PFOA 0.010-0.160 0.080 10 0.021-0.117 0.091 10 <0.008b-0.077 0.036 2 <0.008

b-0.042 0.033 6 <0.097

b-0.184 0.062 5 <0.008

b-0.193 0.050 4

PFNA 0.250-1.04 0.471 10 0.239-0.936 0.484 10 0.189-0.382 0.234 10 0.172-0.388 0.301 10 0.282-1.34 0.578 10 0.272-1.32 0.620 10

PFDA 0.492-1.72 1.17 10 0.275-2.05 0.885 10 0.641-2.26 0.900 10 0.406-1.46 0.912 10 0.350-5.06 2.01 10 2.27-15.1 5.88 10

PFUnA 0.655-2.20 1.28 10 0.463-2.19 0.953 10 0.958-3.15 1.43 10 0.719-2.48 1.45 10 0.633-3.33 1.43 10 1.89-18.4 6.25 10

PFDoA 0.156-0.591 0.362 10 0.073-0.737 0.210 10 0.277-0.949 0.392 10 0.172-0.631 0.371 10 <0.166b-0.810 0.317 9 0.362-3.45 1.01 10

PFTriA 0.173-0.739 0.267 10 0.111-0.528 0.304 10 0.232-0.702 0.370 10 0.162-0.594 0.280 10 <0.070b-0.854 0.259 8 <0.070

b-1.85 0.646 8

PFTA <0.008b-0.131 0.022 8 <0.008

b-0.096 0.039 9 0.031-0.188 0.109 10 0.011-0.148 0.042 10 <0.008

b-0.146 0.029 4 <0.082

b-0.774 0.241 7

PFHxS 0.100-0.308 0.166 10 0.071-0.320 0.119 10 0.080-0.172 0.112 10 0.057-0.303 0.105 10 0.130-0.623 0.445 10 0.099-0.566 0.353 10

PFOS 3.41-10.2 7.13 10 4.21-14.3 7.82 10 1.36-6.23 2.65 10 1.57-4.71 3.81 10 5.89-41.2 16.0 10 4.50-57.0 20.2 10

Lake Apopka (AP)

n = 10

Bear Island (BI)

n = 10

Kiawah Island (KA)

n = 10

Lake Kissimmee (KS)

n = 10

Lochloosa Lake (LO)

n = 10

Merrit Island (MI)

n = 15

St. Johns River (JR) Lake Trafford (TR) WCA-2A (2A) WCA-3A (3A) Lake Woodruff (WO) Yawkey (YK)

n = 10 n = 10 n = 10 n = 10 n = 10 n = 10

an > RL indicates the number of samples above the reporting limit (RL) bValues were calculated with half the RL substituted for nondetects as described in the methods section, but values shown as “<” a

specified number describe the actual RL

NA = Not applicable

Table 2. Correlations between plasma PFAA concentrations and snout-vent length (SVL) for American alligators (Alligator

mississippiensis) sampled in Florida and South Carolina (nmale = 65, nfemale = 60). Refer to table 1 for PFAA abbreviations. Values

were calculated using log normal concentrations. Bold indicates significant correlation coefficients.

SVL PFOA PFNA PFDA PFUnA PFDoA PFTA PFTriA PFHxS PFOS

Male 0.072 0.252a 0.206 0.355b 0.279a 0.209 0.273a 0.273a 0.331b

Female 0.133 0.261a 0.443b 0.489b 0.469b 0.468b 0.570b 0.412b 0.551b a Correlation is significant at the 0.05 level (2-tailed). b Correlation is significant at the 0.01 level (2-tailed).

Table 3. Correlations between concentrations of various PFAAs in plasma of American alligators (Alligator mississippiensis) sampled

in Florida and South Carolina (nmale = 65, nfemale = 60). Refer to table 1 for PFAA abbreviations. Values were calculated using log

normal concentrations. Bold indicates significant correlation coefficients.

Male PFOA PFNA PFDA PFUnA PFDoA PFTriA PFTA PFHxS PFOS

PFOA - 0.615b 0.226 0.092 0.036 0.152 0.181 0.260 0.386

b

PFNA - 0.550b

0.322a

0.144 0.313a

0.273b

0.339b

0.541b

PFDA - 0.840b

0.743b

0.439b

0.654b

0.307a

0.550b

PFUnA - 0.920b

0.783b

0.826b

0.445b

0.654b

PFDoA - 0.751b

0.846b

0.316a

0.528b

PFTriA - 0.770b

0.395b

0.489b

PFTA - 0.238 0.399b

PFHxS - 0.827b

PFOS -

Female PFOA PFNA PFDA PFUnA PFDoA PFTriA PFTA PFHxS PFOS

PFOA - 0.648b

0.332a

0.186 0.098 0.064 -0.003 0.441b

0.440b

PFNA - 0.585b

0.444b

0.365b

0.339b

0.196 0.387b

0.538b

PFDA - 0.890b

0.827b

0.529b

0.560b

0.337b

0.595b

PFUnA - 0.938b

0.684b

0.578b

0.331a

0.691b

PFDoA - 0.763b

0.708b

0.226 0.635b

PFTriA - 0.713b

0.190 0.598b

PFTA - 0.130 0.454b

PFHxS - 0.654b

PFOS - a Correlation is significant at the 0.05 level (2-tailed). b Correlation is significant at the 0.01 level (2-tailed).

Figure 1.

Figure 2.

Figure 3.

A

B

SUPPLEMENTAL INFORMATION

Table S1. American alligator plasma sampling site descriptions

Sampling Location Abbreviation State Coastal vs Inland ⁰N ⁰W Year(s) n

Tom Yawkey Wildlife Center YK SC Costal 33.107 79.132 2014 10

Kiawah Island KA SC Coastal 32.363 80.045 2015 10

Bear Island Wildlife Management Area BI SC Coastal 32.364 80.264 2014 10

Lochloosa Lake LO FL Inland 29.496 82.152 2012 10

Lake Woodruff WO FL Inland 29.107 81.404 2014 10

Lake Apopka AP FL Inland 28.614 81.634 2014 10

Merritt Island National Wildlife Refuge MI FL Coastal 28.523 80.682 2011-2014 15

St. Johns River JR FL Inland 28.196 80.820 2012 10

Lake Kissimmee KS FL Inland 27.905 81.222 2012 10

Lake Trafford TR FL Inland 26.436 81.499 2012 10

Water Conservation Area 2A 2A FL Inland 26.319 80.523 2012 10

Water Conservation Area 3A 3A FL Inland 26.215 80.689 2012 10

Table S2. PFAA concentrations (ng/g) in American alligator (Alligator mississippiensis) plasma

from multiple sites in Florida and South Carolina. Values shown as “<” a specified number

describe the actual reporting limit. SVL = snout-vent lengthRefer to table 1 and figure 1 for

chemical and site abbreviations, respectively. NA = Not available. Collection Site Sex SVL (cm) PFOA PFNA PFDA PFUnA PFDoA PFTriA PFTA PFHxS PFOS

YK F 111.0 <0.099 0.398 4.13 5.72 0.552 <0.070 <0.082 0.356 10.4

YK F 120.0 <0.103 0.439 2.27 3.57 0.525 <0.080 <0.086 0.566 4.50

YK F 131.0 <0.100 0.894 14.3 18.4 2.24 1.04 0.329 0.547 31.6

YK F 135.0 <0.099 0.735 5.55 8.41 1.06 0.624 <0.082 0.510 12.8

YK F 144.8 <0.099 0.272 9.63 12.6 1.79 1.37 0.406 0.470 57.0

YK M 106.7 0.193 1.17 6.20 5.03 0.962 0.668 0.148 0.131 24.6

YK M 165.1 0.014 1.24 7.62 6.77 1.57 1.15 0.241 0.288 50.8

YK M 170.2 0.131 1.32 15.1 11.9 3.45 1.85 0.774 0.351 55.2

YK M 171.5 <0.008 0.286 2.89 2.93 0.672 0.485 0.118 0.099 15.9

YK M NA 0.008 0.506 2.61 1.89 0.362 0.345 0.076 0.107 7.57

KA F 109.2 0.028 0.952 6.59 6.11 6.98 2.60 1.38 0.313 57.2

KA F 119.4 0.105 1.27 6.39 4.05 3.02 0.821 0.432 0.351 38.4

KA F 123.8 0.055 1.14 4.03 3.41 2.91 1.65 0.606 0.374 51.3

KA F 137.4 0.242 1.36 3.72 1.87 1.32 0.420 0.284 1.23 46.9

KA F 141.0 0.129 1.37 6.32 4.06 3.09 1.16 0.689 1.86 98.2

KA M 91.0 0.069 0.446 5.12 3.26 2.51 0.671 0.401 0.499 56.4

KA M 112.4 0.298 0.824 6.52 3.98 3.85 0.857 0.657 1.52 65.0

KA M 132.0 0.218 1.38 13.6 7.53 7.27 1.04 0.198 0.825 74.5

KA M 143.0 0.209 1.02 6.13 3.89 3.26 0.980 0.520 0.742 55.2

KA M 168.0 0.124 1.24 6.21 3.54 1.91 0.550 0.243 0.451 48.1

BI F 108.0 <0.213 0.690 1.18 2.46 0.454 0.674 <0.177 0.824 17.2

BI F 109.0 0.193 1.14 2.01 2.05 0.470 0.414 0.101 0.220 16.1

BI F 134.0 <0.098 0.359 0.998 2.17 0.522 1.02 <0.082 0.411 18.2

BI F 136.6 <0.008 0.155 1.75 2.05 0.653 1.01 0.246 0.101 22.0

BI F 162.2 <0.008 0.462 1.18 1.06 0.259 0.352 0.083 0.077 10.0

BI M 118.0 0.105 0.556 1.20 1.05 0.231 0.244 0.039 0.142 12.2

BI M 128.0 0.089 0.781 3.21 3.22 0.832 0.839 0.202 0.148 24.0

BI M 128.0 <0.097 0.483 2.37 3.74 0.596 <0.070 <0.081 0.388 20.8

BI M 157.6 <0.099 0.371 1.39 2.76 0.795 0.428 0.193 0.440 22.5

BI M 168.0 <0.100 0.339 2.38 5.02 1.88 1.83 0.733 0.510 44.9

AP F 103.8 0.128 0.911 2.03 3.14 0.831 1.00 0.194 0.177 15.8

AP F 111.0 0.143 1.40 2.44 3.39 0.564 0.547 0.050 0.373 13.3

AP F 114.5 <0.096 0.251 0.169 0.614 <0.157 0.189 <0.080 0.379 1.98

AP F 130.5 0.121 1.01 1.15 1.61 0.299 0.441 0.055 0.291 13.2

AP F 144.0 0.152 0.492 0.839 1.25 0.260 0.390 0.047 0.166 9.93

AP M 122.9 <0.098 0.471 0.691 1.68 0.360 0.615 <0.082 0.449 7.26

AP M 136.0 <0.098 0.296 0.520 1.10 0.207 0.464 <0.082 0.388 6.31

AP M 146.0 0.124 0.512 1.09 1.74 0.300 0.416 0.044 0.256 8.14

AP M 162.0 0.140 0.784 1.90 2.38 0.528 0.559 0.091 0.190 12.9

AP M 185.0 0.147 1.08 1.90 1.36 0.330 0.317 0.052 0.435 15.1

LO F 76.0 0.079 0.577 0.543 0.848 0.114 0.213 <0.009 0.069 2.43

LO F 76.1 <0.008 0.328 0.238 0.580 0.105 0.181 <0.008 0.093 2.19

LO F 94.0 0.095 0.720 0.596 0.930 0.120 0.250 <0.008 0.092 2.96

LO F 105.5 0.010 0.561 0.501 1.03 0.218 0.312 0.012 0.104 4.98

LO F 144.0 0.008 0.416 0.425 0.858 0.158 0.352 0.033 0.079 3.02

LO M 95.8 0.132 0.956 0.708 1.04 0.161 0.284 0.019 0.078 4.18

LO M 108.8 0.064 0.891 0.634 1.10 0.202 0.306 0.017 0.105 4.24

LO M 128.4 0.125 1.19 0.996 1.56 0.277 0.471 0.052 0.121 5.18

LO M 159.0 0.073 0.632 0.825 1.33 0.273 0.580 0.060 0.093 4.63

LO M 186.0 0.069 1.15 0.792 1.49 0.309 0.487 0.060 0.201 6.16

WO F 99.0 0.105 0.799 2.16 1.49 0.342 0.192 <0.009 0.234 12.1

WO F 102.0 <0.101 0.282 0.350 0.633 <0.166 0.102 <0.084 0.457 5.89

WO F 103.5 0.074 1.08 3.06 1.98 0.386 0.373 0.049 0.138 19.8

WO F 106.4 <0.101 0.313 0.717 1.20 0.218 <0.070 <0.084 0.471 6.04

WO F 113.0 0.184 1.05 1.84 1.24 0.291 0.272 0.037 0.433 13.5

WO M 58.5 <0.100 0.505 2.03 2.01 0.386 0.854 <0.083 0.530 22.0

WO M 135.0 <0.097 0.482 1.38 1.14 0.224 <0.070 <0.080 0.546 15.8

WO M 157.3 0.152 1.34 5.06 3.33 0.810 0.697 0.146 0.388 41.2

WO M 170.0 <0.096 0.603 2.22 2.34 0.465 0.315 <0.080 0.623 33.7

WO M 171.0 0.087 0.554 1.99 1.36 0.265 0.246 0.020 0.130 16.2

Table S2. (continued) Collection Site Sex SVL (cm) PFOA PFNA PFDA PFUnA PFDoA PFTriA PFTA PFHxS PFOS

MI F 121.0 <0.098 0.410 0.753 1.39 0.203 0.915 <0.082 1.72 43.0

MI F 123.0 <0.096 0.298 0.395 0.844 0.190 0.961 <0.080 3.01 178

MI F 129.0 <0.096 0.611 1.02 1.87 0.276 0.608 <0.080 2.32 62.0

MI F NA 0.115 1.10 2.08 1.92 0.717 0.413 0.152 10.0 206

MI F NA 0.037 0.577 0.816 1.02 0.191 0.197 0.036 3.98 85.5

MI M 135.0 <0.332 0.895 1.66 2.88 <0.543 <0.026 <0.275 2.56 99.5

MI M 135.0 0.235 0.590 0.938 2.64 0.393 0.948 <0.088 1.46 43.2

MI M 136.0 <0.096 0.528 1.20 2.83 0.400 0.674 <0.080 1.50 38.6

MI M 143.0 <0.097 0.855 3.49 5.45 1.07 0.684 0.257 7.33 452

MI M 144.0 <0.098 1.10 1.80 3.31 0.607 0.564 0.098 3.83 118

MI M 152.0 0.306 0.653 0.507 1.04 0.209 1.05 <0.081 4.07 52.6

MI M 154.0 0.144 0.483 0.944 1.38 0.275 0.635 <0.082 4.87 113

MI M 154.0 0.412 0.740 1.33 1.69 0.445 1.42 0.219 4.20 115

MI M 163.0 0.199 0.798 1.40 1.82 0.440 0.474 0.165 23.3 172

MI M 181.0 <0.133 0.347 0.725 1.75 0.435 0.425 <0.110 0.684 38.8

JR F 86.0 0.160 1.04 1.72 2.20 0.432 0.205 <0.008 0.124 9.31

JR F 88.0 0.017 0.369 0.492 0.655 0.156 0.173 0.013 0.308 3.41

JR F 96.0 0.019 0.368 1.20 1.03 0.226 0.203 0.017 0.221 9.23

JR F 126.0 0.083 0.447 0.843 0.903 0.234 0.232 0.023 0.165 4.88

JR F 135.6 0.010 0.494 0.870 1.31 0.591 0.739 0.131 0.219 7.33

JR M 117.5 0.109 0.692 1.14 1.25 0.271 0.321 0.045 0.177 5.25

JR M 126.0 0.082 0.356 1.23 1.31 0.399 0.406 0.081 0.105 7.82

JR M 137.0 0.011 0.603 1.45 1.87 0.441 0.359 0.021 0.166 6.92

JR M 146.0 0.079 0.540 1.51 1.63 0.438 0.447 0.081 0.108 10.2

JR M 168.2 0.082 0.250 0.740 0.994 0.326 0.227 <0.008 0.100 5.76

KS F 90.0 0.113 0.860 1.17 0.841 0.148 0.252 0.028 0.476 7.88

KS F 90.4 0.105 0.275 0.417 0.314 <0.009 0.122 <0.009 1.50 6.51

KS F 106.2 0.115 1.18 2.08 1.73 0.346 0.455 0.085 1.17 13.1

KS F 115.1 0.102 0.542 1.12 0.909 0.114 0.229 0.017 0.719 11.2

KS F 124.0 0.125 0.591 1.32 1.08 0.138 0.229 0.015 0.612 11.3

KS M 96.0 0.079 0.693 0.866 0.760 0.131 0.225 0.022 0.338 7.48

KS M 109.2 0.017 0.463 1.19 0.990 0.147 0.250 0.019 0.493 13.3

KS M 142.5 0.093 0.507 1.53 1.34 0.165 0.295 0.038 0.485 13.3

KS M 160.0 <0.008 0.928 3.15 2.47 0.382 0.677 0.104 0.486 25.1

KS M 167.0 0.142 0.836 2.28 1.72 0.301 0.485 0.079 0.517 20.3

TR F 50.6 0.117 0.555 0.640 0.694 0.148 0.159 0.013 0.176 7.41

TR F 70.5 0.021 0.239 0.275 0.463 0.112 0.111 <0.008 0.127 4.21

TR F 86.9 0.094 0.284 0.424 0.533 0.073 0.169 0.014 0.123 5.30

TR F 93.0 0.067 0.438 0.488 0.619 0.125 0.152 0.018 0.320 5.61

TR F 105.0 0.066 0.458 1.46 2.15 0.737 0.509 0.096 0.123 14.3

TR M 99.0 0.099 0.510 1.13 1.15 0.257 0.331 0.036 0.112 13.4

TR M 109.0 0.087 0.392 0.617 0.753 0.162 0.278 0.035 0.088 5.96

TR M 134.0 0.099 0.803 1.44 1.44 0.296 0.435 0.086 0.091 8.25

TR M 139.9 0.097 0.646 1.66 1.56 0.342 0.440 0.074 0.071 8.23

TR M 142.0 0.022 0.936 2.05 2.19 0.522 0.528 0.055 0.116 10.9

2A F 62.0 <0.008 0.251 0.887 1.29 0.281 0.232 0.056 0.121 3.74

2A F 88.0 0.077 0.237 0.708 1.09 0.409 0.398 0.140 0.080 2.34

2A F 91.4 <0.072 0.189 0.913 1.57 0.489 0.537 0.158 0.172 2.79

2A F 91.8 <0.008 0.207 0.755 1.18 0.374 0.297 0.053 0.101 2.15

2A F 105.0 <0.072 0.216 0.641 0.958 0.277 0.342 0.087 0.085 1.36

2A M 47.5 <0.008 0.362 1.05 1.58 0.324 0.253 0.031 0.120 5.68

2A M 88.0 0.019 0.247 0.733 1.15 0.371 0.308 0.045 0.105 2.51

2A M 130.2 <0.071 0.218 1.10 1.77 0.575 0.574 0.182 0.088 2.37

2A M 132.0 <0.071 0.231 1.25 2.32 0.655 0.655 0.188 0.144 4.79

2A M 140.5 <0.008 0.382 2.26 3.15 0.949 0.702 0.130 0.168 6.23

3A F 78.2 0.031 0.388 1.08 1.80 0.386 0.250 0.011 0.182 4.33

3A F 81.1 <0.008 0.203 0.406 0.881 0.211 0.165 0.015 0.077 2.03

3A F 82.0 0.027 0.361 0.740 1.23 0.300 0.272 0.044 0.127 2.78

3A F 94.3 0.011 0.336 0.858 1.47 0.349 0.428 0.077 0.303 4.52

3A F 99.5 <0.071 0.172 1.08 1.43 0.410 0.288 0.072 0.082 3.98

3A M 94.0 0.040 0.309 0.878 1.15 0.363 0.231 0.033 0.112 3.75

3A M 102.1 <0.008 0.199 0.498 0.719 0.172 0.162 0.018 0.057 1.57

3A M 115.7 0.042 0.286 1.11 1.52 0.378 0.370 0.041 0.060 2.41

3A M 142.0 <0.072 0.293 0.947 1.92 0.599 0.534 0.140 0.106 3.87

3A M 157.0 0.016 0.385 1.46 2.48 0.631 0.594 0.148 0.105 4.71

Table S3. Sex-based differences by site investigated on a site by site basis for PFOA, PFNA,

PFDoA, PFTA, and PFHxS concentrations (log; ng/g) in plasma from American alligators

(Alligator mississippiensis) sampled at multiple sites in Florida and South Carolina Bold

indicates significant correlation coefficients. Refer to table 1 and figure 1 for chemical and site

abbreviations, respectively.

Site PFOA PFNA PFDoA PFTA PFHxS

YK 0.597 0.267 0.917 0.354 0.009b

KA 0.251 0.228 0.602 0.208 0.465

BI 0.675 0.888 0.175 0.584 0.602

LO 0.175 0.008b

0.028a

0.016a

0.175

WO 0.341 0.866 0.251 0.259 0.347

AP 0.753 0.652 0.917 0.625 0.175

MI 0.047a

0.365 0.111 0.637 0.903

JR 0.753 0.752 0.347 0.583 0.076

KS 0.117 0.810 0.251 0.222 0.076

TR 0.463 0.050a

0.117 0.141 0.009b

2A 0.142 0.091 0.175 0.97 0.465

3A 0.465 0.853 0.465 0.402 0.175 a Correlation is significant at the 0.05 level (2-tailed). b Correlation is significant at the 0.01 level (2-tailed).

Table S4. Site differences in PFDA and PFHxS concentrations (ng/g) in plasma of male and

female American alligators (Alligator mississippiensis) sampled at multiple sites in Florida and

South Carolina. Refer to figure 1 for site abbreviations.

Male

Site n

YK 5 C D A B C D E

KA 5 D E F

BI 5 A B B C D E F

LO 5 A A B C

WO 5 B C D E F

AP 5 A B C D E F

MI 10 A B G

JR 5 A B A B C D

KS 5 A B F

TR 5 A B A B

2A 5 A B A B C D

3A 5 A B A

PFDA PFHxS

Female

Site n

YK 5 D C D E

KA 5 D E

BI 5 C A B C D

LO 5 A A

WO 5 C B C D E

AP 5 B C A B C D E

MI 5 A B C F

JR 5 B C A B C D

KS 5 B C E

TR 5 A A B C D

2A 5 A B C A B

3A 5 A B C A B

PFDA PFHxS

Different letters represent statistically significant differences between groups (p < 0.05).

Figure S1. Site PFAA fingerprint for (A) male and (B) female American alligators (Alligator

mississippiensis) sampled in Florida and South Carolina.. Refer to table 1 and figure 1 for

chemical and site abbreviations, respectively.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

YK KA BI LO WO AP MI JR KS TR 2A 3A

PFOS

PFHxS

PFTA

PFTriA

PFDoA

PFUnA

PFDA

PFNA

PFOA

A

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

YK KA BI LO WO AP MI JR KS TR 2A 3A

PFOS

PFHxS

PFTA

PFTriA

PFDoA

PFUnA

PFDA

PFNA

PFOA

B

Figure S2. Mean (±SD) concentrations (ng/g) of (A) PFDA (p = 0.0003), (B) PFUnA (p =

0.021), and (C) PFTriA (p = 0.021) in plasma of American alligators (Alligator mississippiensis)

sampled at multiple sites in Forida and South Carolina.Refer to figure 1 site abbreviations.

0

2

4

6

8

10

12

14

YK KA BI LO WO AP MI JR KS TR 2A 3A

ng/g

pla

sma

Male

Female

A

0

2

4

6

8

10

12

14

16

YK KA BI LO WO AP MI JR KS TR 2A 3A

ng

/g p

lasm

a

B

0

0.5

1

1.5

2

2.5

YK KA BI LO WO AP MI JR KS TR 2A 3A

ng

/g p

lasm

a

C

Figure S3. Signifcant (p < 0.05) site- and sex-based differences in mean (±SD) concentrations of

PFNA, PFTA, PFHxS, PFTA, and PFDoA in plasma of American alligators (Alligator

mississippiensis) sampled at multiple sites in Florida and South Carolina: (A) Lochloosa Lake

(LO) had three PFAAs that showed sex-based differences: PFNA (p = 0.016), PFTA (p = 0.032),

and PFDoA (p = 0.032), (B) the Merritt Island National Wildlife Refuge (MI) site showed sex-

based difference in PFOA burden (p = 0.047), and (C) Lake Trafford (TR) showed sex-based

differnces for PFHxS (p= 0.008), (D) Yawkey (YK) also showed sex-based differences for

PFHxS (p = 0.008), and (E) Lake Trafford (TR) showed sex based-differences in PFNA (p =

0.050). Median and interquartile ranges (error bars) represented in (A) – (E). Refer to table 1 and

figure 1 for chemical and site abbreviations, respectively.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

PFNA PFTA PFDoA

ng/g

pla

sma

Male

Female

A

*

*

*

0

0.05

0.1

0.15

0.2

0.25

ng/g

pla

sma

PFOA

B *

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

ng/g

pla

sma

PFHxS

C *

0

0.1

0.2

0.3

0.4

0.5

0.6

ng/g

pla

sma

PFHxS

D *

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

ng/g

pla

sma

PFNA

*E

Figure S4. Snout-vent length (SVL) of (A) male and (B) female American alligators (Alligator

mississippiensis) sampled at multiple sites in Florida and South Carolina during this study. Refer

to figure 1 for site abbreviations.

Different letters represent statistically significant differences between groups (p < 0.05).